For Time-resolved fluorometry (TRF) applications an optimal luminescent lanthanide chelate has to fulfill several requirements 1) it has to be photochemical stable both in ground and excited state, 2) it has to be kinetically and chemically stable, 3) the excitation wavelength has to be as high as possible, preferable over 300 nm, 4) it has to have efficient cation emission i.e. high luminescence yield i.e. brightness (excitation coefficient x quantum yield, εφ), 5) the luminescence decay time has to be long, 6) the chelate has to have good water solubility, 6) for labeling it should have a reactive group to allow covalent attachment to a biospecific binding reactant, and 7) the affinity and nonspecific binding properties of the labeled biomolecules have to be retained.
Generally, the excited energy transfers from a ligand to a specific lanthanide ion through the ligand's triplet state. However, if the ligand presents a low-energy charge-transfer (CT) state, the sensitization can occur directly from the relaxed CT state without any participation of the triplet (Andraud, C., Maury, O., Eur. J. Inorg. Chem 2009, 4357). Also, excitation directly through the ligand's singlet state has been demonstrated.
Since the publication of label chelates which contain 1-3 separate 4-(phenylethynyl)pyridines (U.S. Pat. No. 4,920,195; Takalo, H., et al., 1996, Helv. Chim.Acta., 79, 789) and 4-phenylpyridines (EP 0195413; WO 87/07955), the designed ligand structures have been applied in many patents, patent applications and publications. One generally used method to improve luminescence intensity is to enhance chelate's molar absorptivity by having several independent chromophoric moieties i.e. 4-(phenylethynyl)pyridines and 4-phenylpyridines combined in structure designs, which offer high stabilities and luminescence quantum yields (see e.g. US 2013/210165 A1 and US 2013/183771 A1).
It is generally known that the luminescence intensity is improved also by increasing chromophore's molar absorptivity together with quantum yield. The molar absorptivity can be enhanced by increasing the it-electron conjugation of the aromatic chromophore. At the same time, the excitation wavelength is normally red shifted. However, the increased conjugation normally decreases the triplet state and/or CT's energy level and decreased quantum yield and decay time are observed. For this reason, in practical bio-label applications the molar absorptivities of the used single chromophores in the lanthanide chelates are in best cases few tens of thousands. Moreover, the increased aromatic conjugation of chromophore decreases the chelate's water solubility, increases unspecific binding properties and formation of aggregates of lanthanide labels as well as the labeled biomolecule. Regarding 4-phenylethynylpyridine and 4-phenylpyridine based chelate labels the higher aromatic conjugation chromophore candidates such as 4-[4-(phenylethynyl)phenylethynyl]pyridine, 4-(4-phenyl-1,3-butadiyne-1,4-diyl)pyridine, 4-(biphenyl-ethynyl)pyridine, 4-biphenylpyridine, 4-(benzoylphenylethynyl)pyridine, naphthylethynylpyridine and 4-(2-fluorenylethynyl)-pyridine (see e.g. Takalo, H., at al. J. Alloys and Compounds, 225(1995)511; D'Aleo, A., et al. Inorg Chem., 47(2008)10269; Picot A., et al. lnorg. Chem. 46(2007)2659) have been published. However, the disclosed chelate designs have not shown any significant improved luminescence intensity compared to the parent basic chelates. For example, Picot et al. describes that for the europium chelate of 4-(2-(7-hexyloxy-9,9′-dihexylfluorenyl)ethynyl)-2,6-bis(diethylcarbamoyl)pyridine.[OTf]3 no significant emission is observed at room temperature” and that “[it is likely that either the CT or the triplet state of the pyridine ligand] at room temperature lies too low in energy to sensitize the europium ion”.
A well-known challenge with chelates and ligands having many chromophores is to find out a suitable structure design, which offers high water solubility and at the same time being inert towards any possible bioprocesses. It is known, that the addition of chromophores decreases the solubility of ligands and chelates in water, increases the formation of bio-specific binding reactant aggregates during the labeling process and non-specific binding properties of labeled biomolecules. Aggregates will produce purification problems and reduced yield of labeled material. Moreover, increased non-specific binding of labeled biomolecule will enhance back-ground luminescence of biospecific assays and thus reduces assay sensitivity.
Contrary to earlier published structures the new chromophoric lanthanide chelate designs have shown improved luminescence intensities i.e. brightness as well as high excitation wavelengths. Those chromophores have shown surprisingly high molar absorptivities the main reason behind the observed luminescence intensities.